The aminoacyl-tRNA synthetases are enzymes with the common general function of catalyzing the following reaction: EQU aaRS+aa+ATP.rarw..fwdarw.aaRS.multidot.aa-AMP+PP.sub.i aaRS.multidot.aa-AMP+tRNA.rarw..fwdarw.aa-tRNA+aaRS+AMP
(aaRS=aminoacyl-tRNA synthetase; aa=amino acid; ATP=adenosine 5'-triphosphate; AMP=adenosine 5'-monophosphate; PP.sub.i =inorganic pyrophosphate) The second (aminoacylation) step is often referred to as "charging" the tRNA.
Generally, in each eucaryotic organism, there are 20 different cytoplasmic aaRSs, one specific for each amino acid. Eucaryotic organisms also generally encode a separate set of mitochondrial aaRSs. It is known that in the yeast Saccharomyces cerevisiae, for example, the cytoplasmic and mitochondrial enzymes are encoded by separate nuclear genes, with the exception of histidyl- and valyl-tRNA synthetases (Natsoulis, G., et al. Cell 46:235-243 (1986); Chatton, B. et al., J. Biol. Chem. 263:52-57 (1988)). Each aminoacyl-tRNA synthetase enzyme recognizes and reacts with a specific amino acid and one or more tRNAs that recognize the codons specific for that amino acid (cognate tRNAs). To a first approximation, the specificity of the aaRS for the amino acid is determined by protein-amino acid interactions, and the specificity of the aaRS for the tRNA is determined by protein-RNA interactions, using different sites on the aaRS.
About 25-30% of patients suffering from one or both of the autoimmune inflammatory myopathies polymyositis and dermatomyositis have been found to produce antibodies against their own essential aminoacyl-tRNA synthetase enzymes. The resulting "antisynthetase syndrome" is characterized by a number of clinical presentations, including interstitial lung disease, arthritis, Raynaud's phenomenon and fevers, along with the muscle weakness, dyspnea and dysphagia of myositis. (See, for example, Targoff, I. N. et al., J. Clin. Invest. 91:2556-2564 (1993)).
Without sufficiently purified human aminoacyl-tRNA synthetases, the process of diagnosing the "antisynthetase syndrome" and identifying the particular antigenic aminoacyl-tRNA synthetase has been cumbersome. One semi-quantitative method available to measure anti-tRNA synthetase antibody in patient serum has been to measure the inhibition of an aminoacylation reaction, testing each one of 20 radioactively labeled amino acids to identify the type of tRNA synthetase. Improved assays would be desirable.
Because the amino acid sequences of the tRNA synthetases have diverged over evolutionary time, significant differences exist between the structures of the tRNA synthetases of humans and of human pathogens. These differences can be exploited by finding inhibitors of aaRS activity which specifically target a tRNA synthetase of a pathogenic organism. By selectively inactivating one or more aminoacyl-tRNA synthetases of a pathogenic organism with a therapeutic substance that minimally affects the corresponding human enzyme, infections by pathogenic organisms can be controlled.
The isolated, recombinant human alanyl-tRNA synthetase can be used in enzymatic activity and other biochemical assays to test substances found to inhibit the alanyl-tRNA synthetase or other tRNA synthetase of a pathogenic organism. The isolated human alanyl-tRNA synthetase can also be used in further studies to model substances with inhibitory activity specific for a tRNA synthetase of a pathogen.
In a second general type of assay, a tester strain is constructed such that it relies upon the function of a human alanyl-tRNA synthetase gene instead of the endogenous host gene of the cell strain. Such tester strains can be used to isolate the in vivo effect of a substance administered to the cells on the human alanyl-tRNA synthetase; that is, to assess by growth rates and other tests whether the substance has any toxicity by its effect specifically on the human alanyl-tRNA synthetase.